The influence of Janthinobacterium sp. strain SLB01 on the cell culture of primmorphs of the sponge Lubomirskia baicalensis

Sponges (phylum Porifera) are ancient, multicellular metazoans. Freshwater sponges (Demosponges, Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal. Over the last years, there have been mass diseases and death of endemic sponges. Previously, the strain Janthinobacterium sp. SLB01 was isolated from the diseased sponge Lubomirskia baicalensis. The studies of the pathogenicity of the strain Janthinobacterium sp. SLB01 for Baikal sponges has not been carried out, therefore we infected experimentally in vitro to determine its pathogenicity by the cell culture of the primmorphs with subsequent isolation, sequencing, and analysis of the genomes. The purpose of the study is to show that the strain Janthinobacterium sp. SLB01 isolated from the diseased sponge L. baicalensis is a pathogen for the cell culture of primmorphs. The bacteria from the infected samples were isolated and identified as strain Janthinobacterium sp. PLB02. A comparative analysis of the genomes of the strains showed that they are practically identical. The genomes of both strains contain genes vioABCDE violacein, flok formation, and strong biofilm, and the type VI secretion system (T6SS), as the primary virulence factor. These bacterial strains based on a comparison of complete genomes showed similarity with strain Janthinobacterium lividum MTR. Isolated strains of Janthinobacterium sp. are pathogens for cell cultures of primmorphs and L. baicalensis sponges. The results of the study will help to expand the understanding of microbial relationships in the development of disease and the death of Baikal sponges.

Genome Analysis of the Janthinobacterium sp. Strain SLB01 from the Diseased Sponge of the Lubomirskia baicalensis

The strain Janthinobacterium sp. SLB01 was isolated from the diseased freshwater sponge Lubomirskia baicalensis (Pallas, 1776) and the draft genome was published previously. The aim of this work is to analyze the genome of the Janthinobacterium sp. SLB01 to search for pathogenicity factors for Baikal sponges. We performed genomic analysis to determine virulence factors, comparing the genome of the strain SLB01 with genomes of other related J. lividum strains from the environment. The strain Janthinobacterium sp. SLB01 contained genes encoding violacein, alpha-amylases, phospholipases, chitinases, collagenases, hemolysin, and a type VI secretion system. In addition, the presence of conservative clusters of genes for the biosynthesis of secondary metabolites of tropodithietic acid and marinocine was found. We present genes for antibiotic resistance, including five genes encoding various lactamases and eight genes for penicillin-binding proteins, which are conserved in all analyzed strains. Major differences were found between the Janthinobacterium sp. SLB01 and J. lividum strains in the spectra of genes for glycosyltransferases and glycoside hydrolases, serine hydrolases, and trypsin-like peptidase, as well as some TonB-dependent siderophore receptors. Thus, the study of the analysis of the genome of the strain SLB01 allows us to conclude that the strain may be one of the pathogens of freshwater sponges.

PCR-screening of bacterial strains isolated from the microbiome of the Lubomirskia baicalensis sponge for the presence of secondary metabolite synthesis genes

The microbial communities of sponges (Porifera) are often a source of natural bioactive metabolites. From the microbiome of the endemic Lubomirskia baicalensis sponge, 35 bacterial strains were isolated and identified using molecular methods. The strains belonged to the phyla Actinobacteria, Firmicutes, and Proteobacteria (classes Alpha- and Betapriteobacteria). To analyze the strains for the presence of genes in the synthesis of secondary metabolites, polyketide synthases (PKS), PCR screening was applied using degenerate primers. Overall, 15 out of 35 strains contained PCR products corresponding in size to a fragment of the ketosynthase domain of the PKS gene cluster. Thus, the proposed method is applicable for rapid screening of the potential ability of microorganisms of different taxonomic groups to produce secondary metabolites. The work contributes to the study of the taxonomic diversity of cultured microorganisms, potential producers of biologically active substances, isolated from the microbiomes of Baikal sponges.

Cooperative Interaction of Janthinobacterium sp. SLB01 and Flavobacterium sp. SLB02 in the Diseased Sponge Lubomirskia baicalensis

Endemic freshwater sponges (demosponges, Lubomirskiidae) dominate in Lake Baikal, Central Siberia, Russia. These sponges are multicellular filter-feeding animals that represent a complex consortium of many species of eukaryotes and prokaryotes. In recent years, mass disease and death of Lubomirskia baicalensis has been a significant problem in Lake Baikal. The etiology and ecology of these events remain unknown. Bacteria from the families Flavobacteriaceae and Oxalobacteraceae dominate the microbiomes of diseased sponges. Both species are opportunistic pathogens common in freshwater ecosystems. The aim of our study was to analyze the genomes of strains Janthinobacterium sp. SLB01 and Flavobacterium sp. SLB02, isolated from diseased sponges to identify the reasons for their joint dominance. Janthinobacterium sp. SLB01 attacks other cells using a type VI secretion system and suppresses gram-positive bacteria with violacein, and regulates its own activity via quorum sensing. It produces floc and strong biofilm by exopolysaccharide biosynthesis and PEP-CTERM/XrtA protein expression. Flavobacterium sp. SLB02 utilizes the fragments of cell walls produced by polysaccharides. These two strains have a marked difference in carbohydrate acquisition. We described a possible means of joint occupation of the ecological niche in the freshwater sponge microbial community. This study expands the understanding of the symbiotic relationship of microorganisms with freshwater Baikal sponges.

Draft Genome Sequence of Flavobacterium sp. Strain SLB02, Isolated from the Diseased Sponge Lubomirskia baicalensis

ABSTRACT There are significant changes in the consortium of microorganisms of freshwater Baikal sponges during their mass death, which began in 2011. The alleged cause of disease is a significant increase in the number of opportunistic microorganisms. Here, we report the draft genome sequence of Flavobacterium sp. strain SLB02.

Microbiome analysis of healthy and diseased sponges Lubomirskia baicalensis by using cell cultures of primmorphs

Endemic sponges (Demosponges, Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal. These freshwater sponges live in symbiosis with diverse eukaryotes and prokaryotes, including chlorophyll-containing microalgae. Within the last 5 years, the incidence of sponge disease and mortality events in Lake Baikal has increased. The etiology and ecology of these events remain unknown, in part because of the lack of models to study sponge-microbe interactions. In this work, we tested the use of primmorph cell cultures of Lubomirskia baicalensis as a tool for investigating the microbiomes of sponges. We infected primmorphs, cultured in vitro, with samples from diseased sponges and observed, by microscopy, disease symptoms, including loss of green symbionts, associated with mass die-off events. Subsequent sequencing of 16S rRNA gene fragments revealed that the microbiome community of healthy sponge and primmorphs formed a group separate from the community of diseased sponges and infected primmorphs. This confirms the suitability of the primmorph cell culture as a model sponge system. We also discovered mass mortality of green symbionts (Chlorophyta) was associated with a shift in the microbial communities of sponges/primmorphs. Microbes in diseased sponges, and infected primmorphs, belonged mainly to the phyla Bacteroidetes and Proteobacteria and these families Flavobacteriaceae, Burkholderiaceae, and Moraxellaceae. Primmorphs cell culture may provide a model to study interactions between these bacteria and their host and elucidate the cause of mass mortality events.

Two new nematode species of the genus Tobrilus Andrássy, 1959 (Nematoda, Triplonchida) from Lake Baikal, Russia

Two new nematode species of the genus Tobrilus Andrássy, 1959 from Lake Baikal are described and illustrated. The first species Tobrilus elginus sp. nov. was found in the littoral zone of Maloye More Bays. The second species Tobrilus juliae sp. nov. dwells on bodies of dead sponges Lubomirskia baicalensis (Pallas, 1776). Tobrilus elginus sp. nov. is most similar to T. amabilis Tsalolikhin, 1974 and T. bekmanae Tsalolikhin, 1975. In contrast to the first species it has a shorter body and spicules, longer gubernaculum and a shorter supplements row. Its body is shorter and thinner, tail and supplement row are shorter and the vulva is more posterior as compared to the second species. The body size of Tobrilus juliae sp. nov. is most similar to T. securus Gagarin & Naumova, 2011 and T. saprophagus Naumova & Gagarin, 2017. From the first of these species it differs by the thinner body, shorter tail, comparatively shorter outer labial setae and shorter spicules. It differs from the second species by a thinner body, shorter male tail and shorter labial setae.

Draft Genome Sequence of Janthinobacterium sp. Strain SLB01, Isolated from the Diseased Sponge Lubomirskia baicalensis

The draft genome sequence of Janthinobacterium sp. strain SLB01, a violacein-producing psychrotolerant bacterium isolated from the diseased sponge Lubomirskia baicalensis, was determined. We identified five genes encoding VioA, VioB, VioC, VioD, and VioE proteins related to violacein biosynthesis that were like those identified in published Janthinobacterium lividum strains MTR and RIT308.